The long-term objectives of this proposal are to understand the molecular interactions of hydrophobic drugs with cell plasma membranes and to establish how such interactions regulate membrane functions. Although drug-membrane interactions are important, little is known about the transbilayer distributions of hydrophobic drugs in plasma membranes of intact cells. Detailed knowledge of drug effects on specific transmembrane signaling processes is also lacking. Ignorance in these areas is largely due to the difficulties of examining molecule-membrane interactions in intack cells and to the lack of techniques for examining topographical distributions of hydrophobic molecules. We have developed planar cell and membrane monolayer techniques that circumvent these difficulties and deficits. Intact human erythrocytes and plasma membranes isolated by planar monolayer techniques provide biolgically relevant and highly purified membrane preparations for examination by biochemical and biophysical methods, including our specialized membrane splitting techniques. The specific aims of this proposal are to examine the structural details of the molecular distribution of the hydrophobic drug tetrahydrocannabinol (THC) and to evaluate membrane- related aspects of phenobarbital (PB) interaction with protein kinase C (PKC). The transbilayer distribution of 3H-THC will be quantified using double-labeled membrane splitting (DBLAMS) and the distribution of the radiolabeled molecule in the plane of the split membrane bilayer determined using monolayer freeze- fracture autoradiography (MONOFARG). The effect of THC on PKC-mediated phosphorylation of membrane proteins wil also be evaluated. The human erythrocyte contains PKC, and phorbol ester tumor promoters stimulate the association of PKC with the membrane and the phosphorylation of membrane proteins. the interaction of 3H-PB with the red cell membrane will be evaluated by single membrane monolayer methods, its transbilayer distribution by DBLAMS, and its in-plane distribution by MONOFARG. The effect of PB on PKC-mediated phosphorylation of membrane proteins will also be evaluated. Understanding the molecular details of membrane interactions of THC will provide insight into the mechanism of psychoactivity of marijuana. Knowledge of the effects of phenobarbital on the transmembrane signaling pathway mediated by protein kinase C will provide insight into the mechanisms of the anticonvulsive and hypnotic properties of the drug.